Summary of Work: We have developed a system to examine the consequences of a site-specific double-strand break (DSB) at a YZ junction in dispensable DNA within the yeast Saccharomyces cerevisiae. A galactose- inducible HO-endonuclease cuts a YZ site placed in a plasmid (YZ-CEN) or at various positions within a YAC that contains human DNA (YAC12). Most site-specific DSBs in the YACs were rapidly repaired and did not lead to arrest or lethality. However, we also identified persistent, long-lived DSBs that can lead to extended G2 arrest and eventual lethality as microcolonies. Examination of different strain backgrounds has suggested differences in the genetic control(s) responsible for indirect lethality from a persistent DSB. We, therefore, utilized a high-copy yeast genomic library to identify clones that convey indirect lethality in response to a persistent DSB in a strain (LS20), that is normally insensitive to the nonrepairable break. We subsequently identified the SIR4 gene as a component in the signaling response. This, as well as SIR2 and SIR3 genes affect transcriptional silencing, chromatin organization, aging and DSB endjoining in yeast. Deletions of any of these genes resulted in enhanced G2 arrest and reduced survival following induction of a single, persistent DSB in a YAC. Based on recent reports, this could be due either to direct effects (at the DSB site) or other effects through loss of the SIR controlled silencing at the silent mating loci, HML or HMR, resulting in a diploid-like phenotype. We, therefore, deleted HML and HMR (by circularizing chromosome III). In this case, the sir3 mutants tolerated the persistant DSB so that cells could proceed more rapidly through the cell cycle (i.e., adapt) and give rise to colonies. Thus, we conclude that turning on the silent HML and HMR loci in the sir3 mutant prevents cells from adapting to the presence of an unrepaired DSB. These results support the hypothesis that the mode of action of SIR gene products in toleration of a DSB is not at the site of a persistent DSB. - Aneuploidy, Cell Extract, DNA Damage, DNA Repair, DNA Recombinant, Genetic Vectors, Mitosis, Plasmids, Saccharomyces Cerevisiae, X-Rays